幽门螺杆菌克拉霉素耐药基因芯片的制备和应用

目的 建立一种寡核苷酸微阵列检测幽门螺杆菌23S rRNA基因A2142G、A2143G及C2182T点突变的方法.方法 根据23S rRNA基因A2142G、A2143G及C2182T突变位点设计相应探针,样本经不对称PCR扩增后,其产物与芯片杂交.非荧光标记引物扩增PCR产物克隆至T载体,测序验证芯片结果,并结合临床最低抑菌浓度实验判断该方法的正确性.结果 寡核苷酸微阵列技术与测序检测幽门螺杆菌23S rRNA基因多态性结果完全一致.经培养及鉴定幽门螺杆菌阳性的54份标本,杂交结果显示A2142位点均为野生型(54/54);A2143G突变率为11.11%(6/54),尚未发现A2143C和A2143T的突变;C2182T突变率为12.96%(7/54),尚未发现C2182A和C2182G的突变,其余均为野生型,上述结果与菌株体外试验MIC结果完全一致.结论 建立一种寡核苷酸微阵列技术检测幽门螺杆菌克拉霉素耐药的23S rRNA基因多态性的方法,可以高通量并直接检测胃黏膜而不需进行细菌培养,推动个体化治疗方案的实施.

An oligonucleotide microarray approach for clarithromycin-resistance Helicobacter pylori detection

Objective To develop an oligonucleotide array to detect single nucleotide mutations in 23S rRNA gene.Methods Primers and probes targeting A2142G.A2143G and C2182T mutations in 23S rRNA gene were designed tp develop an oligonucleotide array.Samples were performed by an asymmetric PCR and the PCR products were hybridized with the specific DNA microarray chips.Non fluorescence-labeled PCR products were cloned into T vectors.The results of oligonucleofide array were confirmed by direct DNA sequencing and evaluated by minimal inhibitory concentration (MIC).Results The results obtained from oligonucleotide microarray were identical to those of direct sequencing.In 54 Helicobacter pylori samples,oligonucleotide microarray indicated that no A-to-C transition at 2142 was found,and the mutant rate of A2143G was 11.11 % (6/54),the mutant rate of C2182T was 12.96% (7/54).A2143C,A2143T,C2182A and C2182G mutations were not found.The other specimens were wild-type.All the above results were the same as that of MIC tests.Conclusions The oligonucleofide microarray is a reliable and accurate genotyping assay for clarithromycin-resistance of Helieobaeter pylofi.It is high-throughput screening method for gastric mucosa and improve the application of strategy for personalized therapy.